Low cost, mass produced point of care (POC) biosensors with sensitive, specific and rapid detection technologies have potentially enormous epidemiological impact on global human and veterinary health. No other societal need would benefit from suitable devices as much as the timely detection and treatment of infectious diseases, especially acute ones, often of epidemic proportions, affecting humans and animals. Suitable POC devices would have to be manufactured at a low cost and in large quantities to allow monitoring of large populations of potentially exposed individuals. These sensors need to be robust enough to be used in adverse conditions such as tropical and subtropical climates and also be simple to use by non-laboratory trained personnel in both resource rich and resource limited setting. In addition, such devices should be single-use, low cost and disposable so as not to be a transmitter of disease. Current state of art POC devices, like lateral flow devices, do not meet this need, because of low sensitivity and/or high variability of results, among other flaws. On the other hand, highly sensitive diagnostic tools such as nucleotide detection require sophisticated separation and processing, making these diagnostic devices difficult in field settings. The laboratory based systems are accurate (both sensitive and specific), but they are not easily transportable or rapid and they require specially trained personnel to operate them. The present invention describes a process that results in a highly sensitive biosensor which can be used as a POC device in any number of settings. These biosensors provide a scalable biocoating on known (as well as on innovative) acoustic wave sensors, making the sensors used in a variety of non-biological settings available for use in POC devices in non-laboratory, e.g., field, settings.
Acoustic waves generated in piezoelectric crystals are well known to be extremely sensitive when applied to the device due to mass and/or viscosity changes, resulting in a change in the frequency and/or phase or amplitude of such acoustic waves before and after application of the mass, which can be electronically measured and correlated to presence of the mass. Hence, they are used as very sensitive chemical or gaseous sensors with the ability to detect mass changes in parts per billion or very small changes in temperature or changes in gas concentrations. However, the use of these devices as biosensors, made from piezoelectrically active materials such as crystals has had limited success either because the applied biological fluid suppresses the generated wave or because the applied films are difficult to manufacture consistently on a large scale, limiting them to resource-rich, sophisticated settings such as research laboratories.
Attempts at functionalizing these acoustic wave sensors as biosensors have resulted in tedious or inconsistent and difficult-to-scale-up processes. In addition, these coating processes must also take into consideration the electrical conductive units attached to these crystals to transmit the acoustic waves and in particular, must not interfere with wave transmission. (Similarly, the crystal structure must be compatible with wave transmission.) Furthermore, the biocoating processes must not attenuate or destroy the acoustic waves. Given these various limitations, progress on biocoating has consistently used a similar approach—namely to attach a chemical agent to the crystal surface which can also bind bioactive agents such as antibodies etc. Two methods are commonly used for depositing the first functional coating: one involves applying a thin layer of vacuum sputtered gold that is reactive with the sulfur function on heterobifunctional thiols or derived disulfides (such as carboxymethyl-Peg-thiol, 5000, Laysan Bio) and the other involves direct functionalization of hydroxyl groups on piezoelectric sensor surfaces with a suitable commercially available heterobifunctional silane, e.g. 3-aminopropyl triethoxysilane (APTES), 3-glycidoxypropyl triethoxysilane (GOPS), 3-mercaptopropyl triethoxysilane (MPTS), to form covalent mono or divalent silicate bonds with the silanes. Another avidin affixation method involves first the deposition of lipids (Annals of Chem. Vol 69:4808-4813) and hydrogels onto a surface acoustic wave sensor (Australian Patent 07473551) followed by deposition of avidin.
These methods are often time consuming and the processes for coating complex. They require prolonged liquid phase contact with a heterobifunctional silane reagent. These silanes are generally provided as solutions in non-reactive solvents like toluene, 2-propanol, or in an aqueous solvents, etc. to deposit mono or multilayers. However, this process is difficult to control for several reasons. First, some of the silanes, such as trimethoxysilanes are very reactive; others, like triethoxysilanes, are less reactive but hydrolytically less stable. Another reason is that because of the reactivity of silanes, their possession of several linkable groups and their tendency to react and link up with additional silane molecules, it is difficult to achieve a single layer of silane deposit which changes the transmission properties of the coated substrate in a manner difficult to control and maintain uniform. The presence of even trace amounts of water additionally complicates process control because the silanes become even more prone to crosslinking by forming reactive silanols or silanediols. The commonly used APTES is particularly prone to multilayer formation. The heterobifunctional silane, GOPS, bears an epoxy or oxirane group that is selectively reactive with thiols, amines and hydroxyl groups, primarily depending on pH, e.g. with thiols at pH of about 7, with amino groups at pH about 9, and with hydroxyl groups at pH>10. It can be used for direct conjugation to one or more amino groups on antibodies or to about half the amino groups on immobilized avidin, such as neutravidin. But these procedures involve wet chemistries and covalent bonds which complicate and delay the coating process and raise the possibility of side reactions, which would interfere with accuracy of the resulting device. (G T Hermanson in Bioconjugate Techniques, 1996, page 142).
In summary, the prior art SAM processes for depositing the intermediary and final protein layer on a piezoelectric material require sequential steps, comprising incubations with multiple reagents in aqueous solvents or inert aprotic media, intermediate rinses, pH changes and final exposure to the binding protein to complete the desired functional SAM affinity biosensor. These stepwise processes may take hours or even several days and may yield SAM biosensors that are functionally variable, expensive and thus unacceptable for POC biosensor applications. Such processes in the art are clearly not suitable for consistent and cost-effective manufacturing of uniform biosensors at low cost and in a scalable high-throughput mode of potentially millions of single use biosensors where process variations or failure would be highly undesirable.
The invention describes, in part, a process which allows direct binding of proteins such as, not only avidins but also other biological materials (nucleic acids, other proteins) having the requisite linkable groups directly on the crystal surface resulting in stable, scalable processes for making uniform, reliable biosensors. The present invention further incorporates the use of both types of sensors, namely, bulk waves and shear horizontal surface acoustic waves and can be used on a number of piezoelectrically active crystal materials. Using this direct coating, the present invention has overcome many of the difficulties mentioned above.
While piezoeletric materials used to create acoustic wave guides are well known and comprise lithium niobate, lithium tantalite, quartz and a few other stances, each provide unique advantages and disadvantages in developing a platform device. Accordingly, it is desirable that any coating method be applicable to all piezoelectric material substrates in order to provide the largest potential variations for detecting the varied and multiple agents in nature responsible for causing infections and their consequences, including but not limited to bacteria, viruses, proteins, nucleotides, parasites, fungus, among others, and notably both individual components thereof and larger particulates such as fragments of pathogens.